Generation of triacylglycerols

ABSTRACT

A method is disclosed for the generation of triacylglycerols from gums that have been separated from an oil product. The gums are treated with an enzyme having PLC activity, which results in the formation of diacylglycerols and phosphates, and treated with an enzyme having PLA activity, which results in the formation of lyso-phospholipids and free fatty acids. The diacylglycerols and the free fatty acids from these two separate reactions then combine in the presence of the enzymes to generate new triacylglycerol molecules.

RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 11/970,270, filed Jan. 7, 2008, now U.S. Pat. No.8,241,876 entitled “GENERATION OF TRIACYLGLYCEROLS FROM GUMS”, thedisclosure of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

This invention relates to a method of generating triacylglycerols fromgums that are recovered from an oil refining process. More particularly,this invention relates to an enzymatic process for the treatment ofvarious phospholipids and lecithins (known collectively as “gums”) fromvegetable oils to produce or “generate” triacylglycerols (triglyceridesor oils). The invention described herein is further work based on theinventions disclosed in U.S. patent application Ser. No. 11/668,921filed Jan. 30, 2007 and U.S. patent application Ser. No. 11/853,339filed Sep. 11, 2007, both of which are assigned to the common assigneeand incorporated herein by reference.

Crude vegetable oils obtained from either pressing or solvent extractionmethods are a complex mixture of triacylglycerols, phospholipids,sterols, tocopherols, free fatty acids, trace metals, and other minorcompounds. It is desirable to remove the phospholipids, free fatty acidsand trace metals in order to produce a quality salad oil with a blandtaste, light color, and a long shelf life. Such removal ofphospholipids, known as “gums,” has been accomplished in the prior artby various methods including water degumming, acid degumming, causticdegumming, and enzyme degumming. Most of these degumming methods involvesignificant loss of oil along with the separated gums.

The aforementioned patent applications disclose methods of removingphospholipids from oil compositions by treating the oil compositionswith both PLA enzymes and PLC enzymes. The treatment with the twoenzymes can be either sequential or simultaneous. It was found that,surprisingly, the kinetics of the enzyme reactions proceed much morerapidly than expected when the two enzymes are used together than wheneither one is used separately. Further, it was found that when the twoenzymes are used together the reactions proceed more rapidly thanexpected even if the reaction conditions are not optimized for at leastone of the enzymes. It also was found that when the two enzymes are usedtogether the reaction can proceed in less than about one hour, and canproceed as quickly as about thirty minutes.

The reactions of PLA and PLC enzymes with the oil compositions areexpected to produce certain reaction byproducts that must be removedfrom the treated oil. These byproducts include phosphate bearingmoieties cleaved from phospholipids by the PLC enzymes, free fatty acidscleaved from phospholipids by the PLA enzymes, and lyso-phospholipidsresulting from the cleavage of the free fatty acid from thephospholipid. The lyso-phospholipids and any phosphate-bearingbyproducts must be removed from the treated oil composition, and it isexpected that the other aforementioned reaction byproducts would beremoved along with the lyso-phospholipids in a heavy fraction known as“gums.”

U.S. Pat. No. 5,061,498 relates to a method for reforming fats and oilswhich comprises treating the fats and oils containing partial glycerideswith two or more different kinds of lipases which are different in afatty acid specificity and/or position specificity in the presence of asmall amount of water to obtain fats and oils containing partialglycerides in a low content. In the disclosed embodiment, lipase P isused because it will react at any of the three positions on a glycerolbackbone. A desired fatty acid such as oleic acid can be added to acomposition containing partial glycerides, and a lipase specific to thatdesired fatty acid such as lipase F is used. The presence of lipase Fpromotes the reaction of the preferred fatty acid over other fatty acidsthat may be present, and the presence of lipase P promotes theesterification of the preferred fatty acid at any location on thepartial glycerides. The water concentration preferably is less than 1500ppm, particularly 10 to 200 ppm.

It is an object of the present invention to provide a method fortreating separated gums to obtain usable oil products that otherwisewould be lost.

SUMMARY OF THE INVENTION

In furtherance of the work described in the two aforementioned patentapplications, analyses were performed of gums that had been separatedfrom PLA/PLC treated oils. It was expected that the gums would containfree fatty acids and diacylglycerols present in an amount proportionalto the amount of phospholipids present in the original oil compositionthat had been reacted upon by the enzymes. Instead it was found that,surprisingly, there were substantially less free fatty acids anddiacylglycerols than would have been expected by theory. From thissurprising result it was concluded that the free fatty acids and thediacylglycerols that were the byproducts of the PLA and PLC reactions,respectively, with the phospholipids had reacted with one another in thepresence of the PLA and PLC enzymes to form useful triacylglycerols,thus actually generating new oil molecules that had not existed beforethe PLA/PLC treatment process commenced. It thereby was discovered thatthe combination of PLA and PLC enzymes could be used to treat separatedphospholipids, regardless of the method used to separate thosephospholipids, to generate new triacylglycerol molecules.

Accordingly, the present invention relates to a method of generatingtriacylglycerols from oil gums, the method comprising (a) providing anoil composition containing a quantity of oil gums, said gums comprisingphospholipids, (b) separating said oil gums from said oil composition toprovide a first fraction substantially free of oil gums and a secondfraction containing said separated oil gums, (c) treating said secondfraction with one or more enzymes having PLA activity to generate freefatty acids, and (d) treating said second fraction with one or moreenzymes having PLC activity to generate diacylglycerols, such that saidfatty acids and said diacylglycerols react with one another in thepresence of at least one of said enzymes to form triacylglycerols.

DESCRIPTION OF THE FIGURES

FIG. 1 is a drawing illustrating the configuration of phospholipid andtriacylglycerol.

FIG. 2 is a drawing illustrating the three sterospecific locations of aphospholipid.

FIG. 3 is a drawing illustrating the structures of four commonfunctional groups that can be attached to the phosphate moiety of aphospholipid.

FIG. 4 is a drawing illustrating four different sites of enzyme attackon a phospholipid molecule.

FIG. 5 is a drawing illustrating the reaction of a phospholipid in thepresence of a PLA enzyme and water to produce a lyso-phospholipid and afatty acid.

FIG. 6 is a drawing illustrating the reaction of a phospholipid in thepresence of a PLC enzyme and water to produce a diacylglycerol and aphosphate.

FIG. 7 is a drawing illustrating the structure of phosphatidyl choline.

FIG. 8 is a drawing illustrating the structure of phospho-choline.

DETAILED DESCRIPTION OF THE INVENTION

The removal of phospholipids generates almost all of the lossesassociated with the refining of vegetable oils. As illustrated in FIG.1, phospholipids contain a phosphate group on one of the two ends of theglycerol backbone, whereas a triacylglycerol contains three fatty acids.In order to differentiate derivatives, the Sterospecific Numbering(“Sn”) system is employed. FIG. 2 depicts the three sterospecificlocations of a phospholipid.

The phosphate group of the phospholipid is “hydrophilic” or“water-loving,” meaning that both the phosphate itself and thefunctional group X are attracted to water. The phospholipid's fatty acidchains R1 and R2 are “lipophilic” or “lipid-loving,” meaning that theyare attracted to lipids. Since the phospholipid molecule possesses botha hydrophilic functional group and lipophilic fatty acid chains, it isan excellent natural emulsifier. The emulsification properties ofphospholipids will cause the removal of two phospholipid molecules andone molecule of triacylglycerol when the phospholipids are removed fromvegetable oils.

The phospholipid's phosphate-containing functional group denoted in FIG.1 as “X” determines the degree of its hydrophilic nature. The functionalgroup X in FIG. 1 may be any of several of a variety of known types, afew of which are illustrated in FIG. 3.

Phospholipids containing the functional groups—choline and—ethanolaminehave the greatest affinity for water, while the acids, acid salts (suchas calcium, magnesium, and iron), and inositol have much loweraffinities for water. Phosphatidic acid and the salts of phosphatidicacid are commonly known as “Non Hydratable Phospholipids” or NHPs.Phospholipids are commonly measured in oil as “phosphorous content” inparts per million. Table 1 sets forth the typical amounts ofphospholipids present in the major oilseed crops, and the distributionof the various functional groups as a percentage of the phospholipidspresent in the oils. Table 2 sets forth the typical distribution ofphospholipids present in lecithin (soybean gums). In Table 2, “as is”means the typical phospholipid composition removed from vegetable oilwith the entrained oil (2 molecules of phospholipids and 1 molecule ofoil), yielding an Acetone Insoluble content of 67%. “Normalized” meansthe phospholipid composition without any oil present, yielding anAcetone Insoluble content of 100%. Table 3 sets forth the molecularweights of the major types of phospholipids, lyso-phospholipids, and thecorresponding non-lipid phospho-compounds. The term lyso-phospholipid asused in Table 3 and throughout this application means a phospholipidthat has had one of its fatty acid groups cleaved by a lipase. Themolecular weight of oleic acid is 282.48, and the molecular weight ofthe diacylglycerol wherein the fatty acids are present as oleic acid(C18:1) is 620.99.

TABLE 1 Typical phospholipid amounts and distributions for oils fromcommon oilseeds. Soy Oil Canola Oil Sunflower Oil P (ppm) 400-1200200-900 300-700 PC (-choline) 12%-46%  25%-40% 29%-52% PE(-ethanolamine) 8%-34% 15%-25% 17%-26% PA (-acid) 2%-21% 10%-20% 15%-30%PI (-inositol) 2%-15%  2%-25% 11%-22%

TABLE 2 Typical phospholipid amounts and distributions for soybean gumsPercentage Percentage “As-Is” “Normalized” Phosphatidyl Choline 33.947.2 Phosphatidyl Ethanolamine 14.3 19.9 Phosphatidyl Serine 0.4 0.6Phosphatidyl Acid 6.4 8.9 Phosphatidyl Inositol 16.8 23.4 Total 71.8100.0

TABLE 3 Molecular weights of typical phospholipids and compounds lyso-Phospho- Phospholipid Phospholipid compound Molecular MolecularMolecular Weight Weight Weight Choline - C18:1 786.15 521.67 183Ethanolamine - C18:1 744.00 479.52 141 Serine - C18:1 787.03 522.55 184Acid - C18:1 721.90 457.42 98 Inositol - C18:1 863.98 599.50 260

Phospholipids can be partially or totally removed from vegetable oilsthrough several different processes, most commonly water degumming, aciddegumming, caustic refining, and enzymatic degumming. The presentinvention of generating oils from gums can be used on gums derived fromany of these processes; for purposes of illustration, enzyme degummingwill be explained in more detail.

Enzymatic degumming, also known as “enzymatic refining,” is used whenthe goal is the total removal of phospholipids from the oil. Generally,enzymatic degumming treatments of the prior art have been practiced onoils that have been degummed previously by one of the other methods,typically water degumming. For food applications, the enzyme degummedoil can be sequentially submitted to bleaching and deodorization, aprocess known in the industry as “physical refining.” Enzymaticdegumming provides a better oil yield than water, acid, or causticdegumming, with improved economic results.

The enzymatic reaction changes the nature of the phospholipid, cleavingdifferent functional groups of the molecule. The functional groups andbreakdown products generally can be referred to as “fatty materials” and“phosphorous containing material.” The enzyme reaction reduces theresulting phospholipids' emulsification properties, so that less oil islost when the gums are separated from the oil, thus saving oil. Enzymesexhibiting activity with phospholipids are commonly called“phospholipases”. The types of phospholipase are based on the positionon the phospholipid molecule at which the enzyme reacts, and are knownas PLAT, PLA2, PLC, and PLD. The positions on the phospholipid moleculeat which the different types of phospholipases react are illustrated inFIG. 4. Phospholipase B is an additional enzyme known in the art. Itremoves the final fatty acid present either in the Sn-1 or Sn-2 position(FIG. 2) of a lyso-phospholipid. A summary of the various phospholipasesand their reaction products is set forth in Table 4.

TABLE 4 Fatty Material Phosphorous Containing Material Phospholipase A1fatty acid lyso-phospholipid Phospholipase A2 fatty acid 2lyso-phospholipid Phospholipase B fatty acid glycerophospholipidPhospholipase C diacylglycerol Phosphate-containing head groupPhospholipase D alcohol phosphatidic acid

Each type of phospholipase has its own rate of reaction and its ownoptimal reaction conditions in terms of pH, water concentration. andtemperature. PLA when used alone generally requires a reaction time ofat least about 4 hours, while PLC when used alone generally requires areaction time of about one hour. It is known that enzymatic treatmentshould occur at a pH less than or equal to 8, in order to minimizeundesirable oil saponification, but PLA has an optimum reaction pH of4.5, while PLC has an optimum reaction pH of 7.0. Each enzyme also hasdifferent thermal tolerances. PLA enzymes will denature at about 50° C.while PLC enzymes will denature at about 65° C.

Sequences of amino acids with phospholipase activity are extensivelyreported in the literature and disclosed in patents, and some of thoseare reported to have activity on phospholipids present in vegetableoils. All this is known in the art.

One commercial PLA1 enzyme product with phospholipase activity isNovozymes' phospholipase A1 Lecitase® Ultra. As described in Novozymes'Application Sheet Oils & Fats#2002-185255-01 and 2002-05894-03, thisproduct can be mixed with degummed oil with a 1-1.5% water citricacid-NaOH buffer at 4.5<pH<7.0 and 40° C.<T<55° C. Under suchconditions, the PLA1 selectively hydrolyzes the fatty acid opposite thephosphate functional group on the glycerol backbone to yield polarlyso-phospholipids and polar fatty acids. As illustrated in FIG. 4, thephospholipid molecule loses one hydrophobic functional group, i.e., thefatty acid, leaving the lyso-phospholipid, which now has a hydrophilicphosphate group and a hydrophilic alcohol group. Now with twohydrophilic sites, the lyso-phospholipid molecule is water soluble, andhas lost its emulsification properties. Thus when the water phase isseparated from the oil phase, the lyso-phospholipid is removed in thewater phase, and does not remove any of the oils with it, while thecleaved fatty acid molecule from the phospholipid remains in the oil. Inprior art processes this fatty acid molecule would be removed in asubsequent deodorization process. The PLA1 degumming process thusreduces refining losses by not removing any neutral oil with thelyso-phospholipids in the water phase, such that the only matter removedis the undesired lyso-phospholipid derived from the originalphospholipid molecule.

The theoretical amount of fatty acids that can be generated by reactinggums with a PLA type enzyme can be calculated by determining the totalamount of phospholipids in the gums, the amount of each type ofphospholipid, and finally the change in molecular weight that occurs inthe conversion of a phospholipid into a lyso-phospholipid for each typeof phospholipid present. The percent phospholipid content can becalculated by multiplying the elemental phosphorous level measured inparts per million by 31 (molecular weight of phosphorous, 30.97) anddividing by 10000. The amounts of each type of phospholipid can becalculated by multiplying the total amount of gums by the normaldistribution of each type of phospholipid known for the particular typeof oil. Finally, the amount of liberated fatty acid can be determinedfrom each type of phospholipid.

For example, for crude soybean oil containing 800 ppm of phosphorouswith a “normalized” phospholipid distribution (Table 2), assuming thefatty acids attached to the phospholipids are oleic acid (C18:1), thefatty acids that are expected to be released can be calculated asfollows:

First the percent of total phospholipid present is calculated.

Total Phospholipids=(800 ppm/1,000,000)×31×100=2.48 percent.

Then the amount of each type of phospholipid present is calculated.

Phosphatidyl Choline=(2.48×47.21)/100=1.17 percent

Phosphatidyl Ethanolamine=(2.48×19.92)/100=0.49 percent

Phosphatidyl Serine=(2.48×0.56)/100=0.01 percent

Phosphatidyl Inositol=(2.48×23.40)/100=0.58 percent

Phosphatidic Acid=(2.48×8.91)/100=0.22 percent

Finally, the amount of fatty acids liberated by the reaction of PLA witheach type of phospholipid in the gums is determined by multiplying theamount of each type of phospholipid times the percentage of free fattyacid (FFA) released, the percentage of fatty acid being calculated aswhat remains after the amount of the lyso-phospholipid is subtractedaway (cf. Table 3), as follows:

FFA from PC=1.17×(1−(521.67/786.15))=0.39 percent

FFA from PE=0.49×(1−(479.52/744.00))=0.18 percent

FFA from PS=0.01×(1−(522.56/787.03))=0.00 percent

FFA from PI=0.58×(1−(599.50/863.98))=0.18 percent

FFA from PA=(0.22×(1−(457.22/721.90)=0.08 percent

Total free fatty acids expected to be generated=0.83 percent

While enzymatic degumming offers significant advantages to oilprocessors, it also poses certain disadvantages. One disadvantage isthat the reaction of the enzyme with the phospholipids can be slow andtime consuming. In particular, the reaction of phospholipase A enzymeswith phospholipids can take many hours, depending on reaction variablessuch as pH, temperature, relative concentrations, and mixing conditions.Such prolonged reaction times can have a significant negative impact onthe overall economic value of enzymatic degumming processes. Because ofthe slowness of the PLA reaction, enzymatic degumming is typicallycarried out on oil compositions that have first been subjected to waterdegumming. Thus, the oil may be degummed twice to obtain a product thathas a phosphorous level low enough for its intended purposes.

It is known in the art that PLC enzymes react with a phospholipid byselectively hydrolyzing the phosphate functional group, as shown in FIG.6. The reaction yields a diacylglycerol (“DAG”) and a phosphatidicgroup. The diacylglycerol molecule no longer has the phosphatefunctional group and does not need to be removed from the oil. Forexample, the reaction of Phosphatidyl Choline (PC), FIG. 7, with a PLCwill yield a DAG and the phosphate functional group illustrated in FIG.8, better known as phospho-choline or “C”. The PLC degumming processreduces the refining loss by retaining the oil-soluble DAG, whileremoving only the water-soluble phosphate functional group. No neutraloil is lost when the water phase is removed because the phospholipid hasbeen destroyed. However, the PLC enzyme does not react with all of thephospholipids present in the oil. Generally, PLC does not react witheither phosphatidic acid (PA) or phosphatidic inositol (PI), illustratedin FIG. 3, although a PI-specific PLC, identified as PI-PLC, is known.Yet both PA and PI are non-hydratable phospholipids that remain in oilafter water degumming. Thus oil that has been treated with PLC as thesole enzyme must be further treated such as with caustic or otherenzymes to remove the residual gums.

The theoretical amount of diacylglycerols generated by the reaction ofgums with a PLC type enzyme can be calculated by determining thepercentage of phospholipids in the oil, the amount of each type ofphospholipid in the type of oil, and finally the change in molecularweight that occurs upon conversion of a phospholipid into a DAG for eachtype of phospholipid present in the crude oil. The percent phospholipidcontent in the oil can be calculated by multiplying the elementalphosphorous level measured in parts per million by 31 (molecular weightof phosphorous, 30.97) and dividing by 10000. The individualphospholipids can be calculated by multiplying the total amount of gumstimes the normal distribution of each type of phospholipid. Finally, theamount of diacylglycerol can be determined that is the reaction productof each type of phospholipid.

For example, for crude soybean oil containing 800 ppm of phosphorouswith a “normalized” phospholipid distribution (Table 2), assuming thefatty acids attached to the phospholipids are oleic acid (C18:1), thediacylglycerols that are expected to be released can be calculated asfollows:

First the percent of each type of phospholipid is calculated, asdescribed above.

Next the percent of each type of diacylglycerol (DAG) liberated by thereaction of PLC with the gums can be determined by multiplying theamount of each type of phospholipid by the percentage of diacylglycerols(Table 3), the amount of DAG being what remains after the amount of thephosphate group is subtracted away, as follows:

DAG from PC=1.17×(1−(165.10/786.15))=0.93 percent

DAG from PE=(0.49×(1−(123.10/744.00)=0.41 percent

DAG from PS=(0.01×(1−(166.08/787.03)=0.01 percent

DAG from PI=(0.58×(1−(243.00/863.98)=0.42 percent

DAG from PA=(0.22×(1−(100.92/721.90)=0.19 percent

Total diacylglycerols generated=1.96 percent

The present invention relates to an enzymatic treatment of phospholipidsand phosphorous-containing oil compositions to generate newtriacylglycerol molecules. The inventor has found that, surprisingly,using a combination of phospholipases having PLA activity and PLCactivity not only will cleave the specific “groups”, but alsore-combines a cleaved fatty acid (FA) from a PLA reaction anddiacylglycerol (DAG) from a PLC reaction to produce a triglyceride, oroil. In particular, a Phospholipase A (PLA) reacts with a phospholipidmolecule yielding a FA and lyso-lecithin, while a Phospholipase C (PLC)reacts with a different phospholipid molecule producing a DAG and aphospho-lecithin. The FA from the PLA reaction and the DAG from the PLCreaction then combine by esterification in the presence of one or moreof the enzymes to produce a new triacylglycerol (TAG) molecule.

The present invention is particularly useful when used to further treatgums that have been removed from a crude oil by methods such as waterrefining, acid refining, or caustic refining, or by enzyme refiningother than by a combination of PLA and PLC enzymes. It is believed thatgums that had been separated by caustic refining would benefit by havingthe pH of the gums adjusted to about 8 or less before proceeding withthe oil generation steps of the present invention.

Oils that can be treated in accordance with the present invention mayinclude but are not limited to the following: canola oil, castor oil,coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil,hempseed oil, linseed oil, mango kernel oil, meadowfoam oil, neat's footoil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil,rapeseed oil, rice bran oil, safflower oil, sasanqua oil, soybean oil,sunflower seed oil, tall oil, tsubaki oil, and vegetable oil, and anycombination of the foregoing.

The phospholipase A enzyme used in the method of the present inventioncan be either a phospholipase A1 enzyme or a phospholipase A2 enzyme.The phospholipase C enzyme used in the present invention can be either aphospholipase C and/or an inositol specific phospholipase C. Manyvarieties of enzymes in the phospholipase A and phospholipase C familiesare available commercially; and it is contemplated that such enzymes andtheir equivalents will be suitable for use in the present invention.

In the method of the invention, the different phospholipases usedtogether in an enzymatic degumming process of the present invention canbe mixed together before being added to the oil to be treated.Alternatively, they can be added to the oil separately, eithersequentially or simultaneously.

The degumming process of the present invention is carried out at a pHbelow about 8, preferable between about 3-7, and most preferably betweenabout 4-5. The pH of the enzyme degumming process can be achieved by theaddition of known buffers. Citric acid and sodium hydroxide are wellknown to be suited to this purpose. Other buffering agents can be usedas needed to adjust the pH under specific reaction conditions.

The temperature of the enzymatic degumming process of the presentinvention can be in the range of about 40-80° C., preferably in therange of about 40-60° C., and more preferably in the range of about45-55° C. It has been found that, surprisingly, under the methods of thepresent invention PLA degumming can proceed at a temperature above itsown optimum of 45° C., and closer to the optimum operating temperatureof PLC, without excessive denaturing.

After the oil generation process has been completed on the gums, and thenewly generated oil has been separated from the gums, the newlygenerated oil can be subjected to further processing steps known in theart such as bleaching or deodorizing, as may be necessary or desirabledepending on the end use for which the newly generated oil product isintended.

Various preferred embodiments of the invention are set forth in theexamples below, along with control examples using conditions of theprior art. In each of the examples below, the overhead mixer was aHeidolph mixer model Elector KG with a flat blade paddle; operated at 90rpm for normal agitation and 350 rpm for vigorous agitation. Thecentrifuge was a De Laval Gyro—Tester installed with “The Bowl Unit” forcontinuous separation. The centrifuge bowl was closed with the plugscrews installed. Shear mixing was accomplished with an Ultra-Turraxhomogenizer SD-45 with a G450 rotor stator at 10,000 rpm. The PLATenzyme was Lecitase® Ultra (lot number LYN05007) sold by Novozymes A/Sof Denmark. The PLC enzyme was Purifine™ (PLC lot number 90BU002A1 or90BU004A1) sold by Verenium Corporation of San Diego, Calif. The amountof phospholipids remaining in the treated oil was measured as ppm P inaccordance with the method of American Oil Chemists' Society OfficialMethod Ca 20-99, “Analysis of Phosphorus in Oil by Inductively CoupledPlasma Optical Emission Spectroscopy.” The Free Fatty Acid (FFA) wasmeasured utilizing the American Oil Chemists' Society Official Method Ca5a-40. Moisture was measured using American Oil Chemists' SocietyOfficial Method Ca 2c-25. Neutral oil was measured using the method setforth in the Appendix below. Acetone-insoluble mater includingphospholipids was measured using American Oil Chemists' Society OfficialMethod Ja 4-46. Acid Value was measured using American Oil Chemists'Society Official Method Ja 6-55. The P-31 NMR procedures and theDiacylglycerol (DAG) measurements by High Performance LiquidChromatography with Evaporative Light Scattering Detector (HPLC-ELSD),were performed by the procedures as reported by Verenium Corporation(then known as Diversa Corporation), “Analytical Profiling of SmallScale Reactions of phospholipase-C mediated Vegetable Oil Degumming,” atthe American Oil Chemists Society 2007 meeting.

Of the following Examples, Examples 1-10 correspond directly to Examples13, 14, 18, 23, 24, 27, 29, 31, 33, and 36 of the aforementioned U.S.patent application Ser. No. 11/853,339 filed Sep. 11, 2007, except thatvalues of the free fatty acids (FFA) and diacylglycerols present in theenzyme degummed oil have been measured by the methods set forth aboveand are included herein.

Example 1

1999.1 grams of crude soybean oil containing 769.5 ppm of phosphorouswas heated to 75-80° C. under normal agitation utilizing an overheadmixer. 2.0 grams of 50% w/w solution of citric acid was added andsheared for 1 minute. The oil underwent normal agitation for 1 hour withan overhead mixer. The oil was allowed to cool with agitation at normalspeed until the oil temperature was 40° C., then 2.4 milliliters of 4molar sodium hydroxide solution was added, and the mixture was shearmixed for 10 seconds. The citric acid and caustic formed a weak bufferwith a pH of 5.0. With the temperature maintained at 40° C., 1.5008grams of Verenium Purifine™ (PLC lipase lot number 90BU002A1) was addedfollowed by 30 grams of de-ionized water and the entire mixture wasshear mixed for 120 seconds. The oil mixture was agitated at normalspeed for 60 minutes. With the temperature maintained at 40° C., 0.2132grams of Novozymes' Lecitase® Ultra (PLA1 lipase lot number LYN05007)was added and the entire mixture was shear mixed for 120 seconds. Theoil mixture was agitated at normal speed for 60 minutes at a temperatureof 40° C. The enzyme treated oil was then centrifuged; and the separatedoil and wet gums were collected. The residual phosphorous in the PLCthen PLA1 sequential degummed oil was 6.5 ppm, FFA was 0.56%, and theDAG was 0.69%.

Example 2

2010.5 grams of crude soybean oil containing 785.1 ppm of phosphorouswas cooled to 60° C. under normal agitation utilizing and overheadmixer. With the temperature maintained at 60° C., 1.5316 grams ofVerenium's Purifine™ (PLC lipase lot number 90BU002A1) and 0.2073 gramsNovozymes' Lecitase® Ultra (PLA1 lipase lot number LYN05007) were addedfollowed by 30 grams of de-ionized water and the entire mixture wasshear mixed for 45 seconds. The oil mixture was agitated at normal speedfor 60 minutes at a temperature of 60° C. The enzyme treated oil wasthen centrifuged; and the separated oil and wet gums were collected. Theresidual phosphorous in the PLC and PLA1 combined enzyme mixture atneutral pH produced a degummed oil with a residual phosphorous of 109.6ppm. The FFA was 0.61% and DAG was 0.74%.

Example 3

2005.3 grams of crude soybean oil containing 742.9 ppm of phosphorouswas heated to 75-80° C. under normal agitation utilizing an overheadmixer. 2.0 grams of 50% w/w solution of citric acid was added andsheared for 1 minute. The oil underwent normal agitation for 1 hour withan overhead mixer. The oil was allowed to cool with agitation at normalspeed until the oil temperature was 60° C., then 2.4 milliliters of 4molar sodium hydroxide solution was added, and the mixture was shearmixed for 10 seconds. The citric acid and caustic formed a weak bufferwith a pH of 5.0. With the temperature maintained at 60° C., 0.7491grams of Verenium's Purifine™ (PLC lipase lot number 90BU002A1) wasadded followed by 60 grams of de-ionized water and the entire mixturewas shear mixed for 45 seconds. The oil mixture was agitated at normalspeed for 60 minutes. With the temperature maintained at 60° C., 0.1220grams of Novozymes' Lecitase® Ultra (PLA1 lipase lot number LYN05007)was added and the entire mixture was shear mixed for 45 seconds. The oilmixture was agitated at normal speed for 60 minutes at a temperature of60° C. The enzyme treated oil was then centrifuged; and the separatedoil and wet gums were collected. The residual phosphorous in the PLCthen PLA1 sequential degummed oil was 2.2 ppm. The FFA was found to be0.58% and the DAG was 0.42%.

Example 4

2002.0 grams of crude soybean oil containing 747.3 ppm of phosphorouswas heated to 75-80° C. under normal agitation utilizing an overheadmixer. 2.0 grams of 50% w/w solution of citric acid was added andsheared for 1 minute. The oil underwent normal agitation for 1 hour withan overhead mixer. The oil was allowed to cool with agitation at normalspeed until the oil temperature was 60° C., then 1.8 milliliters of 4molar sodium hydroxide solution was added, and the mixture was shearmixed for 10 seconds. The citric acid and caustic formed a weak bufferwith a pH of 4.5. With the temperature maintained at 60° C., 2.2194grams of Verenium's Purifine™ (PLC lipase lot number 90BU002A1) followedby 60 grams of de-ionized water were added and the entire mixture wasshear mixed for 120 seconds. The oil mixture was agitated at normalspeed for 15 minutes. With the temperature maintained at 60° C., 0.2198grams of Novozymes' Lecitase® Ultra (PLA1 lipase lot number LYN05007)was added and the entire mixture was shear mixed for 120 seconds. Theoil mixture was agitated at normal speed for 15 minutes at a temperatureof 60° C. The enzyme treated oil was then centrifuged; and the separatedoil and wet gums were collected. The residual phosphorous in the PLC andPLA1 sequential treated degummed oil had a residual phosphorous of 4.6ppm. The FFA was 0.37% and the DAG was 0.42%.

Example 5

2000.8 grams of crude soybean oil containing 747.3 ppm of phosphorouswas heated to 75-80° C. under normal agitation utilizing an overheadmixer. 2.0 grams of 50% w/w solution of citric acid was added andsheared for 1 minute. The oil underwent normal agitation for 1 hour withan overhead mixer. The oil was allowed to cool with agitation at normalspeed until the oil temperature was 50° C., then 1.8 milliliters of 4molar sodium hydroxide solution was added, and the mixture was shearmixed for 10 seconds. The citric acid and caustic formed a weak bufferwith a pH of 4.5. With the temperature maintained at 50° C., 2.2500grams of Verenium's Purifine™ (PLC lipase lot number 90BU002A1) wasadded and 0.2216 grams Novozymes' Lecitase® Ultra (PLA1 lipase lotnumber LYN05007) was added followed by 90 grams of de-ionized water andthe entire mixture was shear mixed for 45 seconds. The oil mixture wasagitated at normal speed for 120 minutes at a temperature of 50° C. Theenzyme treated oil was then centrifuged; and the separated oil and wetgums were collected. The residual phosphorous in the PLC and PLA1combined enzyme mixture produced a degummed oil with a residualphosphorous of 1.8 ppm. The FFA was 0.67% and the DAG was 0.40%.

Example 6

2010.0 grams of crude soybean oil containing 810.8 ppm of phosphorouswas cooled to 50° C. under normal agitation utilizing and overheadmixer. With the temperature maintained at 50° C., 2.2608 grams ofVerenium's Purifine™ (PLC lipase lot number 90BU002A1) followed by 30grams of de-ionized water was added and the entire mixture was shearmixed for 60 seconds. The oil mixture was agitated at normal speed for60 minutes. With the temperature maintained at 50° C., 0.1172 grams ofNovozymes' Lecitase® Ultra (PLA1 lipase lot number LYN05007) was addedand the entire mixture was shear mixed for 60 seconds. The oil mixturewas agitated at normal speed for 60 minutes at a temperature of 50° C.The enzyme treated oil was then centrifuged; and the separated oil andwet gums were collected. The residual phosphorous in the PLC and PLA1sequential treated degummed oil at a neutral pH had a residualphosphorous of 72.6 ppm. The FFA was 0.53% and the DAG was 1.03%.

Example 7

2006.3 grams of crude soybean oil containing 795.3 ppm of phosphorouswas heated to 75-80° C. under normal agitation utilizing an overheadmixer. 2.0 grams of 50% w/w solution of citric acid was added andsheared for 1 minute. The oil underwent normal agitation for 1 hour withan overhead mixer. The oil was allowed to cool with agitation at normalspeed until the oil temperature was 50° C., then 2.4 milliliters of 4molar sodium hydroxide solution was added, and the mixture was shearmixed for 10 seconds. The citric acid and caustic formed a weak bufferwith a pH of 5.0. With the temperature maintained at 50° C., 1.5373grams of Verenium's Purifine™ (PLC lipase lot number 90BU002A1) and0.1168 grams Novozymes' Lecitase® Ultra (PLA1 lipase lot numberLYN05007) was added followed by 90 grams of de-ionized water and theentire mixture was shear mixed for 120 seconds. The oil mixture wasagitated at normal speed for 30 minutes at a temperature of 50° C. Theenzyme treated oil was then centrifuged; and the separated oil and wetgums were collected. The residual phosphorous in the PLC and PLA1combined enzyme mixture at a pH of 5.0 produced a degummed oil with aresidual phosphorous of 1.9 ppm. The FFA was 0.17% and the DAG was0.42%.

Example 8

2003.6 grams of crude soybean oil containing 784.8 ppm of phosphorouswas heated to 75-80° C. under normal agitation utilizing an overheadmixer. 2.0 grams of 50% w/w solution of citric acid was added andsheared for 1 minute. The oil underwent normal agitation for 1 hour withan overhead mixer. The oil was allowed to cool with agitation at normalspeed until the oil temperature was 40° C., then 1.8 milliliters of 4molar sodium hydroxide solution was added, and the mixture was shearmixed for 10 seconds. The citric acid and caustic formed a weak bufferwith a pH of 4.5. With the temperature maintained at 40° C., 1.4603grams of Verenium's Purifine™ (PLC lipase lot number 90BU002A1) and0.1021 grams Novozymes' Lecitase® Ultra (PLA1 lipase lot numberLYN05007) was added followed by 40 grams of de-ionized water and theentire mixture was shear mixed for 120 seconds. The oil mixture wasagitated at normal speed for 120 minutes at a temperature of 40° C. Theenzyme treated oil was then centrifuged; and the separated oil and wetgums were collected. The residual phosphorous in the PLC and PLA1combined enzyme mixture at a pH of 4.5 produced a degummed oil with aresidual phosphorous of 10.7 ppm. The FFA was found to be 0.48% and theDAG was found to be 0.83%.

Example 9

2000.4 grams of crude soybean oil containing 697.7 ppm of phosphorouswas heated to 75-80° C. under normal agitation utilizing an overheadmixer. 2.0 grams of 50% w/w solution of citric acid was added andsheared for 1 minute. The oil underwent normal agitation for 1 hour withan overhead mixer. The oil was allowed to cool with agitation at normalspeed until the oil temperature was 40° C., then 1.8 milliliters of 4molar sodium hydroxide solution was added, and the mixture was shearmixed for 10 seconds. The citric acid and caustic formed a weak bufferwith a pH of 4.5. With the temperature maintained at 40° C., 1.508 gramsof Verenium's Purifine™ (PLC lipase lot number 90BU002A1) and 0.1022grams Novozymes' Lecitase® Ultra (PLA1 lipase lot number LYN05007) wasadded followed by 90 grams of de-ionized water and the entire mixturewas shear mixed for 120 seconds. The oil mixture was agitated at normalspeed for 30 minutes at a temperature of 40° C. The enzyme treated oilwas then centrifuged; and the separated oil and wet gums were collected.The residual phosphorous in the PLC and PLA1 combined enzyme mixture ata pH of 4.5 produced a degummed oil with a residual phosphorous of 2.2ppm. The FFA was 0.20% and the DAG was 0.41%.

Example 10

1999 grams of crude soybean oil containing 695.1 ppm of phosphorous washeated to 75-80° C. under normal agitation utilizing an overhead mixer.2.0 grams of 50% w/w solution of citric acid was added and sheared for 1minute. The oil underwent normal agitation for 1 hour with an overheadmixer. The oil was allowed to cool with agitation at normal speed untilthe oil temperature was 60° C., then 1.8 milliliters of 4 molar sodiumhydroxide solution was added, and the mixture was shear mixed for 10seconds. The citric acid and caustic formed a weak buffer with a pH of4.5. With the temperature maintained at 60° C., 1.5296 grams ofVerenium's Purifine™ (PLC lipase lot number 90BU002A1) and 0.1241 gramsNovozymes' Lecitase® Ultra (PLA1 lipase lot number LYN05007) was addedfollowed by 90 grams of de-ionized water and the entire mixture wasshear mixed for 120 seconds. The oil mixture was agitated at normalspeed for 30 minutes at a temperature of 60° C. The enzyme treated oilwas then centrifuged; and the separated oil and wet gums were collected.The residual phosphorous in the PLC and PLA1 combined enzyme mixture ata pH of 4.5 produced a degummed oil with a residual phosphorous of 5.2ppm. The FFA was found to be 0.36% and the DAG was 0.44%.

The focus of the inventions of the prior cited patent applications wasto develop an enzymatic degumming process having reaction conditionsresulting in the lowest possible residual phosphorous values with theleast amount of processing aids, equipment, and time. Once theexperiments were completed and all of the subsequent analytical testingcompleted, it was surprising to discover that the amount of fatty acidsand diacylglycerols generated in the oils did not match the theoreticallevels that should have been produced. Assuming that all of the PC andPE reacted with the PLC, then it would be expected that the DAG contentwould increase by approximately 1.16 to 1.35 percent, depending on thestarting phospholipid content. Assuming that the PLA1 enzyme reactedwith all of the phospholipids, then it would be expected that the FFAcontent would increase by approximately 0.77 to 0.83 percent, againdepending on the starting phospholipid content. Additionally, if the PLCreacted with all of the PC and PE, then the expected FFA increase due tothe remaining phospholipids reacting with the PLA would be roughly 0.53to 0.59 percent for the above examples. The greatest increase in DAG was0.63 percent with a fatty acid increase of 0.12 (Example 6), both wellbelow the expected levels.

Analyses of the separated heavy phase or “gums” were performed onExamples 1-10 above in order to determine if the gums were hydrated andphysically removed; the extent of each enzyme reaction; and if oneenzyme predominately reacted over the other enzyme. Table 5 is acompilation of phospholipid composition/distribution analysis performedon the separated heavy phase via P-31 NMR, indicating the amount ofunreacted phospholipid, the amount of lyso-phospholipids, and the amountof free phosphates, with all the amounts stated in terms of weightpercent of the sample. (Note: No phospholipid data exists for example 6due to microbial deterioration of the sample.)

TABLE 5 Ex PC PE PI PA l-PC l-PE l-PI l-PA “C” “E” “I” “A” 1 0.00 0.001.73 0.00 1.81 0.22 5.71 7.01 3.82 2.75 0.00 0.81 2 0.00 0.00 0.19 0.001.43 2.27 6.11 4.22 4.56 3.40 0.00 0.82 3 1.02 0.00 2.77 0.00 9.55 0.218.30 13.25 2.47 1.34 0.00 0.50 4 0.99 0.00 4.76 0.95 5.28 0.32 5.9310.81 3.73 1.81 0.00 0.49 5 1.62 0.00 3.35 0.78 12.29 0.21 8.28 11.911.49 0.89 0.00 0.46 6 — — — — — — — — — — — — 7 0.00 0.16 1.10 0.00 1.320.38 5.35 1.24 3.56 2.95 0.00 0.93 8 0.00 0.15 1.08 0.00 1.60 0.67 6.371.98 4.37 3.57 0.00 1.33 9 0.08 0.00 3.03 0.00 1.93 0.08 5.07 10.80 3.852.58 0.00 0.84 10 2.09 0.00 4.26 0.00 7.22 0.24 6.90 12.81 2.06 1.140.00 0.49

Example 1 was a sequential addition of enzymes at a pH of 5 allowing thePC and PE to react first with the PLC enzyme, and then allowing the PI,PA, and any remaining PC and PE to react with the PLAT enzyme. The PLCenzyme was in contact with the oil for 60 minutes at 40° C. before thePLA enzyme was added, thus allowing the enzyme to react with all of thePC and PE present in the oil without competition with the PLA enzyme.After the initial PLC reaction, the PLA was added such that the PLAcould hydrolyze the remaining phospholipids present in the oil. Theresidual phosphorous, as noted above, was successfully reduced to 6.5ppm. The starting oil had a DAG content of 0.40 percent and a FFA of0.41 percent compared to the final oil containing 0.69 percent DAG and aFFA of 0.56 percent. If both of the enzymes reacted with the specificphospholipids, then the DAG should have been increased by 1.29 percentto 1.69 percent and the FFA should have increased by 0.55 percent to0.96 percent. The actual DAG was found to have only increased by 0.29percent and the FA only increased by 0.15 percent. P-31 NMR analysis ofthe gums showed all of the original phospholipids were hydrolyzed exceptthe PI. Significant amounts of lyso-PI and lyso-PA were present withminor amounts of lyso-PC and lyso-PE. The phospho-species of “C”, “E”,were also present in the recovered gums, as well as phospho-species “A”,which was a surprise because it has been reported that PLC does notreact with PA. Additionally, no phospho-inositol was detected. Comparingthe distribution found in the gums to the original oil, 1.0 percent DAGand 0.40 percent FA were missing!

Example 2 was a simultaneous enzyme addition, at a pH of 7, with 1.5percent water, and a 45 second shear mix. Both enzymes were in contactwith the oil for 60 minutes at 60° C., thus the enzymes were incompetition with one another. The centrifuged oil had a residualphosphorous of 109.6 ppm; DAG increased 0.34 percent, and the FFAincreased 0.20 percent. The P31-NMR analysis of the collected gumsdetected only a small amount of PI present, and no PC, PE, or PA. Thus,all of the original phospholipids were reacted. Significant amounts ofthe lyso- and phospho-species were detected in the gums, except for “I”.It was surprising to discover that the DAG increased only slightlycompared with Example 1, but the amount of “C” and “E” were greater thanwere found in Example 1.

Example 3 was a sequential enzyme addition, at a pH of 5, with 3 percentwater, and applied shear mixing for 45 seconds after the addition ofeach enzyme at 60° C. The PLC enzyme was in contact with the oil for 60minutes before the PLA enzyme was added and both enzymes were allowed toreact for an additional 60 minutes. The centrifuged oil contained 2.2ppm phosphorous; the DAG essentially did not increase (0.40 to 0.42percent) and the FFA increased only by 0.17 percent. Comparing thecollected gums from Example 3 to Example 2, a large increase in thelyso-species was present while the phospho-species were all depressed.These results indicate that at these conditions, the PLA reactiondominated over the PLC reaction, even though PLC was added first. Theamount of DAG and FFA should have been far greater than was present inthe finished oil based on the disappearance of the startingphospholipids and the appearance of the reaction products!

Example 4 was a sequential enzyme addition, at a pH of 4.5, 3 percentwater, and shear mixing for 120 seconds after the addition of eachenzyme at 60° C. The PLC enzyme was in contact with the oil for 15minutes before the PLA enzyme was added and both enzymes were allowed toreact for an additional 15 minutes. The centrifuged oil contained only4.6 ppm phosphorous, the DAG increased only by 0.02 percent, while theFFA went down by 0.04 percent. An evaluation of the collected gumsshowed some PC, PA, and PI that were not hydrolyzed. The lyso- andphospho-species were not as high as were found in Example 3, but werestill elevated considering the limited contact with the enzymes.

Example 5 was a simultaneous enzyme addition, at a pH of 4.5, with 4.5percent water, and a 45 second shear mix. Both enzymes were in contactwith the oil for 120 minutes at 50° C., thus the enzymes were incompetition with one another during the entire reaction. The centrifugedoil had a residual phosphorous of 1.8 ppm; DAG did not increase at all,and the FFA increased 0.26 percent. The analyzed gums showed a largeincrease in the amount of lyso-species and a decrease in phospho-speciescompared to the previous four examples, indicating that PLA isdominating over PLC in the reaction mixture.

Example 6 was a sequential addition of enzymes at a pH of 7. The PLCenzyme was in contact with the oil for 60 minutes at 50° C. before thePLA enzyme was added, thus allowing the enzyme to react with all of thePC and PE present in the oil without competition with the PLA enzyme.After the initial PLC reaction, the PLA was added such that the PLAcould hydrolyze the remaining phospholipids present in the oil. Theresidual phosphorous, as reported previously, was only reduced to 72.6ppm. The starting oil had a DAG content of 0.40 percent and a FFA of0.41 percent, while the final oil contained 1.03 percent DAG and a FFAof 0.53 percent. If both of the enzymes reacted with the specificphospholipids, then the DAG should have been increased by 1.36 percentto 1.76 percent and the FFA should have increased by 0.57 percent to atotal of 0.98. The actual DAG was found to have increased by 0.63percent and the FFA only increased by 0.12 percent. No P-31 NMR analysisof the gums was available due to microbial deterioration of the sample.

Example 7 was a simultaneous enzyme addition. The enzymes were incontact with the oil sample for a total of only 30 minutes at a pH of 5with 4.5 percent water, and at a temperature of 50° C. The residualphosphorous in the oil was only 1.9 ppm. The lyso-species were alldepressed, especially the lyso-PC. The phospho-species were all twice asmuch as were found in the Example 5 gum analysis, indicating that underthese conditions the PLC dominated over the PLA. However, the DAGcontent essentially did not increase (0.40 to 0.41 percent) compared tothe starting oil and the FFA did not increase to the expected total of0.98 percent, but actually decreased by 0.24 percent for a total FFA of0.17 percent!

Example 8 was a simultaneous enzyme addition, at a pH of 4.5, with 2percent water, and a 120 second shear mix. Both enzymes were in contactwith the oil for 120 minutes at 40° C., thus the enzymes were incompetition with one another during the entire reaction. The centrifugedoil had a residual phosphorous of 13.3 ppm; DAG increased by 0.43, andthe FFA increased 0.07 percent. The lyso-species were all depressedwhile phospho-species were still higher than those in Example 7.

Example 9 was similar to the reaction in Example 8, but instead of 2percent water, 4.5 percent water was added in the simultaneous enzymereaction and the enzyme contact time was 30 minutes instead of 120minutes. The residual phosphorous in the oil was 2.2 ppm. The amount ofDAG present in the oil essentially remained the same as the starting oilwhile the FFA decreased from the initial oil by 0.21 percent. The lowresidual phosphorous level and elevated amounts of all the lyso-species,especially the lyso-PA, indicated high PLA enzyme activity. From this itwas expected that a very large amount of FFA should have been generatedin the reaction process. The phospho-species were slightly depressed incomparison to Example 8, but the DAG should have been significantlyhigher according to the amounts of phospho-species present in the gums.

Example 10 was carried out with a simultaneous enzyme addition, at a pHof 4.5, with 4.5 percent water, and a shear mix of 120 seconds. Theenzymes were in contact with the oil for 30 minutes at 60° C. Theresidual phosphorous in the oil was 5.2 ppm. The amount of DAG presentin the oil essentially remained the same as the starting oil (0.40 to0.44) while the FFA decreased from the initial oil by 0.05 percent;while maintaining roughly the same amount of by-products in the gums asexample 3. The reaction conditions of examples 3 and 10 were different,yet the results in terms of resulting phospho-species, DAG and FFA wereabout the same, indicating that the reaction is very robust in formingTAG.

Table 6 below is a summary of the initial phosphorous, DAG, and FFA ofthe starting oils for each of examples 1-10 above, the theoreticalamount of DAG and FFA that would have been present in the treated oil ifall the phospholipids in the starting oil had reacted with the enzymes,and the measured amounts of phosphorous, DAG and FFA in the treated oil.

TABLE 6 Theoretical Amounts in Measured Amounts in Ex- Starting OilTreated Oil Treated Oil am- Phos DAG FFA DAG* FFA** Phos DAG FFA ple(ppm) (%) (%) (%) (%) (ppm) (%) (%) 1 769.5 0.40 0.41 1.69 0.96 6.5 0.690.56 2 785.1 0.40 0.41 1.71 0.97 109.6 0.74 0.61 3 742.9 0.40 0.41 1.640.94 2.2 0.42 0.58 4 747.3 0.40 0.41 1.65 0.94 4.6 0.42 0.37 5 747.30.40 0.41 1.65 0.94 1.8 0.40 0.67 6 810.8 0.40 0.41 1.76 0.98 72.6 1.030.53 7 795.3 0.40 0.41 1.73 0.98 1.9 0.42 0.17 8 783.9 0.40 0.41 1.710.97 13.3 0.83 0.48 9 697.7 0.40 0.41 1.57 0.91 2.2 0.41 0.20 10 695.10.40 0.41 1.56 0.90 5.2 0.44 0.36 *Theoretical DAG only includesgeneration from phosphatidyl choline and phosphatidyl ethanolamine.**Theoretical FFA only includes the generation from phosphatidyl serine,phosphatidyl inositol, and phosphatidic acid.

P-31 NMR analysis of the gums confirmed that the gums were not beinghydrated and physically separated from the oil as in the “normal” waterand/or acid degumming process, but were hydrolyzed by the PLC and PLAenzymes. The analysis confirmed the formation of the cleavedphospho-species and creation of lyso-lecithins (Table 5). It did not,however, explain why the levels of DAG and FFA were depressed in thetreated oils. No information in the prior art could be found to describewhy significant amounts of DAG and FFA appeared to be missing.

U.S. patent application Ser. No. 11/668,921, and U.S. patent applicationSer. No. 11/853,339 of Dayton et al. disclose an enzymatic method forremoving various phospholipids from vegetable oils to produce a degummedoil with a combination of enzymes in which the reaction period can beone hour or less. The inventors reveal a synergistic effect between PLCenzymes and PLA enzymes, improving the kinetics of the reaction from twoto six hours when the enzymes are used alone to one hour or even lesswhen the two enzymes are used together.

The present invention is based on the unexpectedly low levels of DAG andFFA found in these subsequent analyses of these treated oils. Based onthese results, the invention herein lies in the discovery that PLA andPLC apparently interact synergistically in a matrix of lipids containingphospholipids and their PLA/PLC hydrolysis byproducts to producetriacylglycerols. Without wishing to be bound by theory, it is believedthat the cleaved diacylglycerol byproducts from the PLC hydrolysis andthe cleaved fatty acids from the PLA hydrolysis combine under theenzymatic degumming process conditions to create new triacylglycerols.It is theorized that the proximity or the orientation, or both, of thetwo enzymes allows the formation of the triacylglycerols during therelease of both the diacylglycerols and fatty acids in the water phaseof the reaction.

An additional series of examples was performed on gums produced fromcrude soybean oil utilizing the traditional water degumming process asdescribed in the prior art earlier. The wet gums were obtained directlyfrom an industrial water degumming process. The wet gums were utilizedas a raw material to eliminate the majority of the triacylglycerolspresent in oil while maintaining all the other minor components that maybe present in the “typical” degumming matrix. The P-31 NMR analysis ofthe gums detected only the phosphatidyl species and none of the lyso- orphospho-species. The phospholipid compositional data is listed below inTable 7. The diacylglycerol content of the gums obtained from theindustrial water degumming process was 1.5 percent.

Two control examples were conducted at each of the enzyme's optimumreaction conditions to determine the base case for further analysis ofthe experiments. The first control was at a neutral pH for thephospholipase C enzyme and the second experiment was conducted at a pHof 4.5, the optimum for the phospholipase A enzyme.

Example 11

Control: Phospholipase C (PLC) at neutral pH-50 grams of wet soybeangums were added to a 500 ml round bottom flask. 10 grams of Verenium'sPurifine™ (PLC lipase lot number 90BU004A1) were added. The material wasmixed with an overhead paddle mixer equipped with a stainless steelrounded paddle to fit the curvature of the flask at a rate ofapproximately 150 rpm. The flask was covered with Parafilm® to preventevaporation of the water. The wet gums and enzyme were heated to 45° C.under continuous agitation. The system was maintained for eight hours.The apparatus was then disassembled and the hydrolyzed gums werecollected. The gums were placed in a centrifuge tube and centrifuged for15 minutes at 5000 rpm to separate the light phase (“oil”) from theheavy phase (“gums”).

The DAG content of the recovered oil was determined to be 32.6 percent,compared to a starting DAG of 1.5 percent, for a difference of 31.1percent. The large increase in DAG content is consistent with a PLCreaction, in which the DAG does not react further. The phospholipidprofile obtained by P31-NMR analysis of the heavy phase confirmed thatthe phosphatidyl groups had been hydrolyzed to the phospho-groups.Unexpectedly, a small amount of “I” was detected, as well as smallamounts of all the lyso-groups. Thus the PLC does possess some PLAactivity under the reaction conditions of this example.

Example 12

Control: Phospholipase A (PLA) at pH 4.5—50 grams of wet soybean gumswere added to a 500 ml round bottom flask. 2.0 grams of 50% w/w solutionof citric acid was added and mixed for 5 minutes. Then 1.8 millilitersof 4 molar sodium hydroxide solution was added, and the mixture wasmixed for an additional 5 minutes. The citric acid and caustic formed aweak buffer with a pH of 4.5. 2 grams of Novozymes' Lecitase® (PLATlipase lot number LYN05007) was added. The material was mixed with anoverhead paddle mixer equipped with a stainless steel rounded paddle tofit the curvature of the flask at a rate of approximately 150 rpm. Theflask was covered with Parafilm® to prevent evaporation of the water.The wet gums and enzymes were heated to 45° C. under continuousagitation. The system was maintained for eight hours. The apparatus wasthen disassembled and the hydrolyzed gums were collected. The gums wereplaced in a centrifuge tube and centrifuged for 15 minutes at 5000 rpmto separate the light phase (“oil”) from the heavy phase (“gums”).

The DAG content of the recovered oil was determined to contain 3.8%, anincrease of 2.3 percent. The phospholipid profile obtained by P31-NMRanalysis of the heavy phase confirmed the hydrolysis of the phosphatidylgroups to the corresponding lyso-groups, which is consistent with a PLAreaction. Very small amounts of “C” and “E” were detected, as well the“A”. The PLA does not possess any significant PLC activity.

Example 13

PLA at neutral pH—50 grams of wet soybean gums, obtained from anindustrial degumming centrifuge, were added to a 500 ml round bottomflask. 2 grams of Novozymes' Lecitase® (PLA1 lipase lot number LYN05007)were added. The material was mixed with an overhead paddle mixerequipped with a stainless steel rounded paddle to fit the curvature ofthe flask at a rate of approximately 150 rpm. The flask was covered withParafilm® to prevent evaporation of the water. The wet gums and enzymewere heated to 45° C. under continuous agitation. The system wasmaintained for eight hours. The apparatus was then disassembled and thehydrolyzed gums were collected. The gums were placed in a centrifugetube and centrifuged for 15 minutes at 5000 rpm to separate the lightphase (“oil”) from the heavy phase (“gums”). The DAG content of therecovered oil was determined to contain 2.6 percent, an increase of only1.1 percent DAG. The phospholipid profile showed all of the “original”gums were hydrolyzed, but a depressed amount of lyso- andphospho-derivatives were found in comparison to the control conditionsin Example 12. This suggests that under the reaction conditions of thisexample, the PLA enzyme does not generate DAG, oil, or phospho speciesbut does generate lyso-species and fatty acids.

Example 14

PLC and PLA at neutral pH-50 grams of wet soybean gums, obtained from anindustrial degumming centrifuge, were added to a 500 ml round bottomflask. 10 grams of Verenium's Purifine™ (PLC lipase lot number90BU004A1) and 2 grams of Novozymes' Lecitase® (PLA1 lipase lot numberLYN05007) were added. The material was mixed with an overhead paddlemixer equipped with a stainless steel rounded paddle to fit thecurvature of the flask at a rate of approximately 150 rpm. The flask wascovered with Parafilm® to prevent evaporation of the water. The wet gumsand enzymes were heated to 45° C. under continuous agitation. The systemwas maintained for eight hours. The apparatus was then disassembled andthe hydrolyzed gums were collected. The gums were placed in a centrifugetube and centrifuged for 15 minutes at 5000 rpm to separate the lightphase (“oil”) from the heavy phase (“gums”).

The DAG content of the recovered oil was determined to contain only 7.8percent, compared to the 32.6% DAG content obtained when PLC was usedalone. The phospholipid profile confirmed that all of the phosphatidylgroups where hydrolyzed to the phospho- and lyso-groups. Thephospho-groups of “C”, “E”, and “A” were detected in roughly the sameamount as was detected in Example 11, except that the amount of “I” wasslightly depressed. The l-PE was slightly depressed while l-PC and l-PAwere both roughly twice the amount found in Example 11, but not a largeincrease. The amount of l-PI and l-PA where significantly higher thanwere found in Example 11, since PLA was also in the reaction matrix andconverted the PI and PA to their lyso-forms.

The P-31 NMR analysis not only confirmed the PLC conversion ofapproximately the same amount of phospholipids to their phospho-groupsas in the Control Example 11, but also confirmed that the remainingphosphatidyl groups were converted to their lyso-forms, indicating PLAconversion. This is a surprising result because the pH conditions werenot optimum for PLA conversion. The amount of DAG present in the oilshould have been roughly 33 percent, not 7.8 percent as was determinedby HPLC analysis!

Example 15

PLC and PLA at pH 4.5—50 grams of wet soybean gums, obtained from anindustrial degumming centrifuge, were added to a 500 ml round bottomflask. 2.0 grams of 50% w/w solution of citric acid was added and mixedfor 5 minutes. Then 1.8 milliliters of 4 molar sodium hydroxide solutionwas added, and the mixture was mixed for an additional 5 minutes. Thecitric acid and caustic formed a weak buffer with a pH of 4.5. 10 gramsof Verenium's Purifine™ (PLC lipase lot number 90BU004A1) and 2 grams ofNovozymes' Lecitase® (PLAT lipase lot number LYN05007) were added. Thematerial was mixed with an overhead paddle mixer equipped with astainless steel rounded paddle to fit the curvature of the flask at arate of approximately 150 rpm. The flask was covered with Parafilm® toprevent evaporation of the water. The wet gums and enzymes were heatedto 45° C. under continuous agitation. The system was maintained foreight hours. The apparatus was then disassembled and the hydrolyzed gumswere collected. The gums were placed in a centrifuge tube andcentrifuged for 15 minutes at 5000 rpm to separate the light phase(“oil”) from the heavy phase (“gums”).

The DAG content of the recovered oil was the same as in Example 14, 7.8percent. The phospholipid profile confirmed that all of the phosphatidylgroups where completely hydrolyzed to the phospho- and lyso-groups as inExamples 12 through 14. The phospho-groups of “C”, “E”, “I” and “A” weredetected in roughly the same amount as was detected in Example 11. l-PCand l-PE were significantly higher than were found in Example 11. LikeExample 13, the amount of l-PI and l-PA were significantly higher thanwere found in Example 11, since PLA was also in the reaction matrix andconverted the PI and PA to their lyso-forms. As in the degummingexamples 1 through 10, the amount of DAG actually found was less thanwhat had been expected based on the P-31 NMR analysis; suggesting thatthe DAG and FFA that were generated were consumed in the subsequentgeneration of TAG.

The P-31 NMR analysis not only confirmed the PLC conversion ofapproximately the same amount of phospholipids to their phospho-groupsas in the Control Example 11, but also confirmed that the remainingphosphatidyl groups were converted to their lyso-forms by PLA. Theamount of DAG present in the oil should have been roughly 33 percent,not 7.8 percent as was determined by HPLC analysis!

Table 7 summarizes the phospholipid profiles obtained by P31 NMR, withall numbers stated as weight percent of the heavy phases separated fromthe reaction mixtures of examples 11-15, showing the unreactedphosphatidyl moieties, the lyso-groups generated by PLA conversion, andthe phospho groups generated by PLC conversion.

TABLE 7 Example PC PE PI PA l-PC l-PE l-PI l-PA “C” “E” “I” “A” Starting9.36 9.32 5.50 4.86 0.62 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Material 110.00 0.00 5.45 0.54 0.59 0.30 1.58 1.65 4.27 3.20 0.40 1.36 12 0.00 0.000.00 0.00 19.91 19.01 13.19 10.80 0.05 0.05 0.00 0.39 13 0.00 0.00 0.000.00 7.59 7.17 5.07 3.97 0.12 0.05 0.00 0.17 14 0.00 0.00 0.00 0.00 1.230.23 7.39 3.70 4.40 3.40 0.22 1.47 15 0.00 0.00 0.00 0.00 1.09 2.71 8.294.86 4.25 3.00 0.50 1.37

Table 8 below is a summary of the initial DAG, and Acid Value (AV) ofthe starting gums for each of examples 11-15 above, the theoreticalamount of DAG and FFA that would have been present in the finished oilif all the phospholipids in the starting oil had reacted with theenzymes and the actual DAG present in the final oil. The final FFA wasnot measured because the FFA measurement procedure required more oilthan was available from these experiments. In each of these examples,less DAG was found in the recovered oil than was expected, furthersupporting the conclusion that the DAG was consumed in the generation ofTAG by the reaction of DAG with FFA.

TABLE 8 Starting Theoretical Recovered Ex- Lecithin Total Oil am- DAGAV*** Enzyme DAG* FFA** DAG FFA**** ple (%) (%) Type (%) (%) (%) (%) 111.5 21 PLC 40.2 0.0 32.6 — 12 1.5 21 PLA 1.5 24.1 3.8 — 13 1.5 21 PLA1.5 24.1 2.6 — 14 1.5 21 PLC/PLA 40.2 7.6 7.8 — 15 1.5 21 PLC/PLA 40.27.6 7.8 — *Theoretical DAG only includes generation from phosphatidylcholine and phosphatidyl ethanolamine. **Theoretical FFA includes thegeneration from all phospholipids when PLA is reacted. When PLC and PLAare reacted together, only the FFA from phosphatidyl serine,phosphatidyl inositol, and phosphatidic acid are calculated. ***The AcidValue (AV) is the number of milligrams of potassium hydroxide necessaryto neutralize the acids in one gram of sample (1). AV is arepresentation of the titratable acidity contributed by both thephospholipids and the free fatty acids. (2). ****The FFA was notmeasured due to the large amount of sample required to perform thetitration.

The following Example 16 is identical in terms of process steps toExample 15, other than being doubled in scale. The purpose of thisExample was to perform a mass balance on the entire sample before andafter the PLA/PLC enzyme reaction to verify the generation oftriacylglycerols from the reaction byproducts of the PLA/PLC hydrolysisof the phospholipids.

Example 16

PLC and PLA at pH 4.5—100 grams of wet soybean gums, obtained from anindustrial degumming centrifuge, were added to a 500 ml round bottomflask. 4.0 grams of 50% w/w solution of citric acid was added and mixedfor 5 minutes. Then 3.6 milliliters of 4 molar sodium hydroxide solutionwas added, and the mixture was mixed for an additional 5 minutes. Thecitric acid and caustic formed a weak buffer with a pH of 4.5. 20 gramsof Verenium's Purifine™ (PLC lipase lot number 90BU004A1) and 4 grams ofNovozymes' Lecitase® (PLAT lipase lot number LYN05007) were added. Thematerial was mixed with an overhead paddle mixer equipped with astainless steel rounded paddle to fit the curvature of the flask at arate of approximately 150 rpm. The flask was covered with Parafilm® toprevent evaporation of the water. The wet gums and enzymes were heatedto 45° C. under continuous agitation. The system was maintained foreight hours. Samples of both the starting gums and the enzymaticallytreated mixture were analyzed “as is” for moisture, percent gums, andneutral oil content. Neutral oil content was measured by the method setforth in the Appendix below. The separated neutral oil was then analyzedfor diacylglycerol content. The results are listed in Table 9.

TABLE 9 Starting Gums Enzyme Treated Gums Moisture (%) 24.0 32.7 PercentGums (“as is”) 55.8 27.3 Percent Gums (“dry”) 73.4 41.4 Percent NeutralOil (“as is”) 20.2 38.7 Percent Neutral Oil (“dry”) 26.5 58.6 DAG (%)1.5 13.2

The starting wet gums analysis is typical for wet gums obtained from anindustrial water degumming operation on soybean oil. Of the non-aqueousmaterial in the sample, the gums were roughly 73 percent and the neutraloil was roughly 27 percent. The analysis of the “enzyme treated gums”from Example 16 demonstrates that a substantial portion of thephospholipids present were hydrolyzed by the PLA/PLC enzymesphospholipids, as shown by the decrease of gums from 73 to 41 percent,while the amount of triacylglycerols showed an increase from 26.5% to58.6%. Theoretically, the amount of diacylglycerols expected to begenerated by the process was 40.2%, but was found to be only of 13.2%.It is concluded that the combination of PLC and PLA enzymes utilized foroil degumming, purification, or modification of lecithins generatestriacylglycerols, or oil.

There has been disclosed a method of generating triacylglycerols fromphosphatidyl-containing oil gums by treating the gums with a combinationof PLA and PLC enzymes, whereby the DAG by product of the PLC reactionand the FFA byproduct of the PLA reaction combine with one another inthe presence of the enzymes to form new TAG molecules. The two differentenzymes can be reacted with the gums either simultaneously orsequentially; when a sequential process is used, the enzymes can beadded in either order. The reaction time of the enzymes with the gumscan be on the order of about four hours or less, and can be as low asabout thirty minutes. The enzymes having PLA activity can be selectedfrom the group consisting of a phospholipase A1 enzyme and aphospholipase A2 enzyme. The PLA enzyme can be present in the reactionmixture in a concentration of about 2 ppm of active enzyme or less; or 1ppm of active enzyme or less; or as low as 0.5 ppm of active enzyme orless. The enzymes having PLC activity can be selected from the groupconsisting of a phospholipase C enzyme and a phosphatidyl-inositolspecific phospholipase C enzyme. The PLC enzyme can be present in thereaction mixture in a concentration of about 30 ppm of active enzyme orless; or 20 ppm of active enzyme or less; or as low as 10 ppm of activeenzyme or less.

The enzyme reactions can be carried out at a temperature in the range ofabout 40-80° C., preferably in the range of about 40-60° C. The pH canbe in the range of about 3-7. The enzyme reaction can be facilitated byshear mixing, preferably for about forty-five seconds or more whenperformed on a laboratory scale. It is expected that the time allottedfor shear mixing will increase as the process is scaled up to industriallevels, as would be known to those skilled in the art. Also, acetoneprecipitation of the phosphorous containing material would allow therecovery of the generated oil; such a process is known in the art in theproduction of de-oiled lecithins.

While preferred embodiments of the invention have been set forth hereinas known at the time of this application, other embodiments encompassingthe inventive method will be readily apparent to those skilled in theart, and all such embodiments and their equivalent are intended to becovered by this application encompassed by the claims hereof.

APPENDIX

The following method was used to determine the neutral oil of theexamples in this application.

Definition

This method determines the total neutral oil found in wet gums,lyso-gums, or crude oil soapstock.

Scope

Applicable to gums, lyso-gums, and soapstock.

Reference

A.O.C.S. Method G 5-40

A.O.C.S. Method Ca 2c-55

A.O.C.S. Method Ja 4-46

Apparatus

1. Graduated Cylinder—100 ml

2. Graduated Cylinder—50 ml

3. Graduated Cylinder—25 ml

4. Disposable Centrifuge Tubes—50 ml (Polypropylene)

5. Separatory Funnel—500 ml

6. Beaker—500 ml

7. Beaker—400 ml

8. Beaker—250 ml

9. Glass Stirring Rods

10. Centrifuge

11. Desiccator

12. Steam Bath

13. Oven—105° C.

Reagents

1. Aqueous potassium hydroxide (KOH)—14% by weight.

2. Sodium Chloride (NaCl)—Reagent grade.

3. Ethyl alcohol—SDA Formulas 30 and 3A are permitted, 50% by volume.Mix 10 volumes of 95% alcohol with 9 volumes of distilled water.

4. Ethyl alcohol—SDA Formulas 30 and 3A are permitted, 10% by volume.Mix 2 volumes of 95% alcohol with 17 volumes of distilled water.

5. Petroleum ether—ACS grade.

6. Acetone—ACS grade.

7. Deionized or distilled water

8. Nitrogen—clean and dry

Procedure

1. Perform % moisture on the sample immediately after the sample ispulled. Note: AOCS 2c-55, temperature is lowered to 105° C. due tofoaming of soap samples at 130° C. Time is increased to 4 hours.

2. Mix sample thoroughly and weigh immediately.

3. Weigh 5 grams (to the nearest 0.0001 g) of sample into a previouslyweighed 50 ml disposable centrifuge tube. (Note: include the cap andbeaker (for holding the centrifuge on scale)).

4. Add 35 ml of cold acetone (kept in an ice bath) to the sample and mixvery well with a glass stir rod. Break up the lecithin precipitate withthe glass rod. Note: The acetone will become bright yellow.

5. Cap the centrifuge tube.

6. Centrifuge the acetone for 5 minutes to separate the gums from theacetone.

7. Pour the acetone into a 250 ml beaker.

8. Repeat steps 4 through 7 four times.

-   -   a. After the last extraction, remove the gums and place into a        previously weighed disposable drying pan. Allow the excess        acetone to evaporate.    -   b. Place sample into a 105° C. draft oven overnight.    -   c. Cool to room temperature in a desiccator and weigh the        contents of the drying pan and sample.    -   d. Calculate the percent gums in the original sample and on a        dry basis.

9. Pour the acetone layer into a 500 ml separatory funnel (“A”).

10. Add 50 ml of water to separatory funnel. Mix.

11. Add 50 ml of Petroleum Ether (P.E.). Mix.

12. Add a pinch of NaCl (˜¼ table spoon of table salt) to the separatoryfunnel. Mix

13. Allow the two layers to separate. Remove the bottom layer(acetone/water), including any emulsion and add it to a new separatoryfunnel (“B-1”). DO NOT DISCARD THE P.E. LAYER.

14. Add 50 ml of Petroleum Ether (P.E.) to the acetone/water from step13. Mix

15. Allow the two layers to separate. Remove the bottom layer(acetone/water), including any emulsion to a new separatory funnel(“B-2”).

16. Add the Petroleum Ether layer to the original P.E. extract from step13, separatory funnel “A”.

17. Repeat steps 14 through 16 two times. The acetone/water layer may beadded to a used funnel “B-1” from above. Once the last extraction iscomplete, you may discard the acetone/water layer.

18. Add 100 ml of 50% ethanol to the separatory funnel containing theP.E. Mix.

19. Add 10 ml of 14% KOH. Mix gently.

20. Add 50 ml of 50% ethanol to separatory funnel. Mix.

21. Allow the layers to completely separate. Do not allow the P.E. layerto remain in contact with the alcohol/KOH layer longer than 30 minutes.DO NOT DISCARD THE P.E. LAYER.

22. Remove the alcohol/KOH layer and place in a new separatory funnel.

23. Add 50 ml of P.E. to the separatory funnel containing thealcohol/KOH layer. Mix.

24. Allow layers to separate. Collect the alcohol/KOH layer into a newseparatory funnel. Add the P.E. layer to the P.E. from step 21.

25. Repeat steps 23 and 24.

26. In the separatory funnel containing the P.E. Layers. Add 25 ml of10% alcohol, shake vigorously. Allow the layers to separate. Remove thealcohol layer. Discard alcohol layer.

27. Repeat step 26 twice. To the third “washing” (alcohol layer), addtwo drops of phenolphthalein to determine if the layer is neutral. Ifthe layer turns pink, repeat step 26.

28. Draw the P.E. layer into a tared beaker that has been previouslydried and cooled in a desiccator. Evaporate the P.E. on a steam bathunder a gentle stream of nitrogen.

29. Once the P.E. has been removed, Place beaker in an oven at 105° C.for 30 minutes.

30. Cool in a desiccator to ambient temperature and weigh.

31. Repeat until constant weight is attained. (A constant weight isattained when the loss (or gain) in weight does not exceed 0.1% insuccessive 30 minute drying periods.)

CalculationNeutral Oil, % (as is)=Mass of Neutral Oil/Mass of Sample×100Neutral Oil, % (dry basis)={Mass of Neutral Oil/Mass ofSample}/{100-Percent Moisture}×100Gums, % (as is)=Mass of Dried Gums/Mass of Sample×100Gums, % (dry basis)={Mass of Dried Gums/Mass of Sample}{100-PercentMoisture}×100

What is claimed is:
 1. A method of generating triacylglycerolscomprising (a) providing an oil composition containing a quantity of oilgums, wherein the gums comprise phospholipids, (b) treating the oilcomposition with one or more enzymes having PLA activity to generatefree fatty acids, (c) treating the oil composition with one or moreenzymes having PLC activity to generate diacylglycerols, (d) reactingthe fatty acids with the diacylglycerols in the presence of at least oneof the enzymes having PLA or PLC activity, thereby formingtriacylglycerols.
 2. The method of claim 1, wherein steps (b) and (c)occur substantially simultaneously.
 3. The method of claim 1, whereinstep (b) occurs before step (c).
 4. The method of claim 1, wherein step(c) occurs before step (b).
 5. The method of claim 1, wherein the totalduration of the treating of the enzymes with the oil composition insteps (b) and (c) and the reacting in step (d) is no more than aboutfour hours.
 6. The method of claim 5, wherein the total duration of thetreating of the enzymes with the oil composition in steps (b) and (c)and the reacting in step (d) is about thirty minutes.
 7. The method ofclaim 1, wherein the one or more enzymes having PLA activity areselected from the group consisting of a phospholipase A1 enzyme and aphospholipase A2 enzyme.
 8. The method of claim 1, wherein the one ormore enzymes having PLC activity are selected from the group consistingof a phospholipase C enzyme and a phosphatidyl-inositol specificphospholipase C enzyme.
 9. The method of claim 1, wherein the treatingof steps (b) or (c) or the reacting of step (d) comprises treating orreacting at a pH of about 8 or less.
 10. The method of claim 9, whereinthe pH is about 3-7.
 11. The method of claim 1, wherein the treating ofthe enzymes with the oil composition in steps (b) and (c) and thereacting in step (d) occurs at a temperature of about 40-80° C.
 12. Themethod of claim 11, wherein the treating of the enzymes with the oilcomposition in steps (b) and (c) and the reacting in step (d) occurs ata temperature of about 40-60° C.
 13. The method of claim 1, wherein theoil composition comprises a crude oil.
 14. The method of claim 1,wherein in steps (c) or (d), the PLC enzyme is present in a quantity ofabout 30 ppm of active enzyme or less.
 15. The method of claim 14,wherein the PLC enzyme is present in a quantity of about 20 ppm ofactive enzyme or less.
 16. The method of claim 15, wherein the PLCenzyme is present in a quantity of about 10 ppm of active enzyme orless.
 17. The method of claim 1, wherein in steps (b) or (d), the PLAenzyme is present in a quantity of about 2 ppm of active enzyme or less.18. The method of claim 17, wherein the PLA enzyme is present in aquantity of about 1 ppm of active enzyme or less.
 19. The method ofclaim 18, wherein the PLA enzyme is present in a quantity of about 0.5ppm of active enzyme or less.
 20. The method of claim 1, wherein thetreating of steps (b) or (c) comprise shear mixing the oil compositionand the respective PLA or PLC enzymes to obtain a mixture.
 21. Themethod of claim 20, wherein the shear mixing continues for a duration ofat least 45 seconds.
 22. The method of claim 20, wherein the treating ofsteps (b) or (c) further comprises adding a quantity of water to themixture.
 23. The method of claim 22, wherein the quantity of water is atleast about 1.5% by weight of the mixture.
 24. The method of claim 23,wherein the quantity of water is at least about 3.0% by weight of themixture.
 25. The method of claim 24, wherein the quantity of water is atleast about 4.5% by weight of the mixture.